Introduction The nucleocapsid (NC) protein of all orthoretroviruses contains strictly conserved Zn2+-finger structures that are necessary for its function in viral replication. NC function is being studied through site directed mutagenesis of these proteins and the Zn2+-finger structures. This Section has determined that NC plays important roles in the various replication steps from assembly processes to assistance with reverse transcription of the viral RNA and also integration processes that link the newly synthesized viral DNA to the cell's chromosomal DNA. By altering the NC protein either chemically or by site directed mutagenesis, we have found that the above mentioned processes can be severely affected. This provides researchers with an attractive target for antiviral interventions as well as new and unique strategies for vaccine development. Significant Achievements Time-course quantitation of reverse transcription intermediates in cells infected with NC mutant HIV-1. The recently published study (J. Virol. 77:1469-1480) has been instrumental in showing NC's role in reverse transcription and integration processes in the cell. Cultures of HOS cells were infected with NC mutant or wild-type HIV-1 and reverse transcription intermediates were quantitated after 24 hrs of incubation with cells. Mutations to NC result in lower levels of reverse transcripts and they appear to be degraded at the ends. This year, these studies have been extended to examine viral DNA production over time. The previous conclusions have essentially been confirmed as the kinetic pattern observed over time for the NCH23C mutant was most likely a consequence of progressive degradation based on lack of protection of the vDNA from nucleases by the mutant NC protein. Additional mutant NC proteins are currently being examined to determine how different mutations in NC affect reverse transcription processes. Carbonyl mutations in the HIV-1 NC Zn2+-finger. Among the more striking features in NC is the conservation of a carbonyl containing amino acid (Asp, Asn, Glu, or Gln) that precedes the COOH-terminal Cys residue of virtually every NC Zn2+-finger. We wanted to determine whether substitutions of these residues could support viral replication. Thus substitutions, summarized in Table 1, were made in the NH2- and COOH-terminal NC Zn2+-fingers of HIV-1 (Asn 27 and Asp 48, respectively - numbering refers to the amino acid position in NC). Variations in amino acid side chains include a small size hydrophobic residue (Gly), a bulky hydrophobic residue (Val), an even bulkier hydrophobic residue (Ile), an amide/acid exchange, and an amide/amide or acid/acid exchange (for the NH2- and COOH-terminal fingers, respectively). Mutations were incorporated into the pNL4-3 proviral plasmid and transfected into 293T cells. Transiently expressed virus was collected and analyzed for RT activities and virion infectivity. Table 1. Substitutions of carbonyl containing amino acids in the Zn2+-fingers of HIV-1. Zn2+-finger NH2-terminal COOH-terminal Asn27Gly Asp48Gly Asn27Val Asp48Val Asn27Ile Asp48Ile Asn27Asp Asp48Asn Asn27Gln Asp48Glu RT activities obtained 72 h after a typical transfection show some variations due to DNA quality and transfection anomalies but for the most part, virus production is not significantly affected. Single round- and long-term infectivity assays have been performed to examine the ability of these mutants to infect cells. Mutant and wild-type viruses were evaluated in these assays. All of the mutants except for Asp48Ile were demonstrably infectious in the single-round assay. It appears that the Asp48Ile mutant is quite defective in its ability to infect cells. Long-term replication assays in H9 cells showed that not only the Asp48Ile, but the Asp48Val mutants are defective: no virus spread was observed by measuring RT activity, even in the undiluted samples. The remaining mutants were replication competent with titers similar to those obtained for the wild-type virus, which could be diluted 1:100,000 and replication still proceeds. Thus even though the Asp48Val mutant is infectious in the single round infection analysis, it is defective for replication over multiple rounds. Thus far, it appears that bulky, hydrophobic amino acid side chains are not well tolerated in the COOH-terminal NC Zn2+-finger of HIV-1. We have also examined the reverse transcription intermediates from cells infected with the two replication defective mutant viruses. Viral DNA isolated from these infected cells 24 h post-infection were similar to wild-type virus in quantity, thus there does not appear to be any reverse transcription defect evident. There may be defects in integration processes in the Asp48Ile and Asp48Val mutants which will be confirmed through further studies. Characterization of cellular nucleic acid binding protein Zn2+-finger substitutions in the HIV-1 NC protein. The study of these NC mutants has been completed. Final studies included examining reverse transcription intermediates from the one replication defective mutant (CNBP-5). It was determined that initiation of reverse transcription was defective with this mutant virus when tested for infection compared to wild-type virus. This study has led to a publication in the Journal of Virology (77:8524-31). Studies using recombinant NC mutant proteins to probe the molecular properties of NC. In collaboration with Drs. Frederic Bushman (Salk Institute), Karin Musier-Forsyth (University of Minnesota), Robert Bambara (University of Rochester Medical Center), and Jeffrey DeStefano (University of Maryland), recombinant NC proteins (mutant and wild type) have been studied to examine the molecular interactions of NC with other viral proteins (e.g. RT and IN) as well as nucleic acids. These studies have resulted in published works from all of the above mentioned laboratories (J. Virol. 77:1598-1603, J. Biol. Chem. 278:30755-63, Nucleic Acids Res. 31:4847-55, J. Biol. Chem. 278:31536-46). Additional studies have been initiated with the DeStefano laboratory. The study published by our laboratories (J. Biol. Chem. 278:30755-63), which involved mutation of groups of amino acid changes, raised additional questions regarding examining the amino acid determinants for nucleic acid secondary structure melting and strand annealing. Therefore we will be examining other NC mutants that mutate these residues individually to determine which ones are important for the nucleic acid melting and annealing processes. We have also started to investigate additional NC properties in collaboration with Gilles Mirambeau (Institut Gustave Roussy, Villejuif, France). Structures called G-quadruplexes are being studied that are important for lentiviral replication, which are located in the central polypurine tract. It is thought that NC may be interacting with these G-quadruplexes, thus compacting the newly made viral DNA so that it may traverse the nuclear membrane prior to integration.